Development and application of technology platform for identification of the hist

历史鉴定技术平台的开发与应用

• 批准号: 7434194
• 负责人: Doug Chan
• 金额: $ 9.74万
• 依托单位: PROTECH LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-19 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7434194
• 关键词: Acetylation; Affect; Affinity; Affinity Chromatography; Amino Acids; Antibodies; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Cell physiology; Chromatin Structure; Code; Condition; Contract Services; Core Protein; Cultured Cells; DNA; DNA Methylation; DNA Sequence; Data; Development; Diagnosis; Disease; Enzymes; Epigenetic Process; Gene Expression; Histone Code; Histone H3; Histones; Iceberg; Industry; Knowledge; Label; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modification; Molecular; N-terminal; Peptides; Phase; Play; Post-Translational Protein Processing; Protein Binding; Protein Methyltransferases; Proteins; Proteomics; Reader; Reagent; Recombinant Proteins; Recruitment Activity; Regulation; Role; Signal Transduction; Site; Stable Isotope Labeling; Tail; Technology; Therapeutic Intervention; Transcriptional Regulation; Tumor Suppressor Proteins; base; combinatorial; drug development; gene function; metaplastic cell transformation; new technology

DESCRIPTION (provided by applicant): Post-translational modifications of the N-terminal tail of histones play important roles in epigenetic regulation. The histone code hypothesis proposes that modifications of histone tails create combinatorial binding sites to recruit enzymes for transcriptional regulation. Dysregulation of histone modifications has been linked with diverse diseases including cancer. While much is known about histone modifications and the modifying enzymes, it remain largely unclear how the modified residues transmit the information to regulate gene expression. Identifications of modification-specific binding proteins will be the first step to fill this knowledge gap. Loss of acetylation of Lys16 (H4K16ac) and trimethylation of Lys20 of H4 (H4K20me3) has been correlated with cancer, but the molecular mechanism is not clear. The "binders" to H4K16ac and H4K20me3 are not known, hindering our understanding of how loss of these modifications may contribute to cancer. We propose to first develop a proteomic platform to identify specific histone modification binders and then use the platform to identify proteins that bind H4K16ac and H4K20me3. Our proteomics platform consists of efficient protein affinity purification with modified histone peptides with SILAC labeling and nanoHPLC-MS/MS for protein identification and quantification. We have preliminary data demonstrating the feasibility of such a platform using histone H3 peptides with modifications at Lys4 and/or Lys9. This Phase I proposal will allow us to: (1) develop a technology platform that will enable us to identify and quantify proteins that bind to any modifications on histone proteins; and (2) identify proteins that bind to H4K16ac and H3K20me3, that may have tumor suppressor functions. Our commercial activities will include: (1) contract services to academic and industry labs for the identification of histone code effectors; and (2) development of a Histone Code Reader Toolkit(r), which consists of recombinant proteins and antibodies that we find to bind modified histones to facilitate functional analysis of the histone code in epigenetics field.

描述（由申请人提供）：组蛋白N-末端尾部的翻译后修饰在表观遗传调控中起重要作用。组蛋白密码假说提出，组蛋白尾部的修饰创造了组合结合位点，以招募酶进行转录调控。组蛋白修饰的失调与包括癌症在内的多种疾病有关。虽然对组蛋白修饰和修饰酶有很多了解，但很大程度上仍不清楚修饰的残基如何传递信息以调节基因表达。修饰特异性结合蛋白的鉴定将是填补这一知识空白的第一步。H4 K16 ac和H4 K20 me 3的乙酰化缺失和三甲基化与肿瘤的发生有关，但其分子机制尚不清楚。H4 K16 ac和H4 K20 me 3的“结合物”是未知的，这阻碍了我们对这些修饰的丢失如何导致癌症的理解。我们建议首先开发一个蛋白质组学平台来识别特定的组蛋白修饰结合剂，然后使用该平台来识别结合H4 K16 ac和H4 K20 me 3的蛋白质。我们的蛋白质组学平台包括使用SILAC标记的修饰组蛋白肽进行高效蛋白质亲和纯化，以及用于蛋白质鉴定和定量的nanoHPLC-MS/MS。我们有初步的数据证明了这样一个平台的可行性，使用组蛋白H3肽与修饰在Lys 4和/或Lys 9。第一阶段的提案将使我们能够：（1）开发一个技术平台，使我们能够识别和量化与组蛋白蛋白上的任何修饰结合的蛋白质;（2）识别与H4 K16 ac和H3 K20 me 3结合的蛋白质，这些蛋白质可能具有肿瘤抑制功能。我们的商业活动将包括：（1）为学术和工业实验室提供合同服务，以识别组蛋白密码效应子;以及（2）开发组蛋白密码阅读器工具包（r），该工具包由重组蛋白和抗体组成，我们发现这些重组蛋白和抗体可以结合修饰的组蛋白，以促进表观遗传学领域组蛋白密码的功能分析。